Transcriptome analysis reveals genes associated with late blight resistance in potato.

Late blight is a serious disease of potato worldwide. Our study aimed to unveil genes involved in late blight resistance in potato by RNA-seq analysis after artificial inoculation under controlled conditions. In this study, two potato somatic hybrids (P7 and Crd6) and three varieties such as Kufri Girdhari, Kufri Jyoti and Kufri Bahar (control) were used. Transcriptiome analysis revealed statistically significant (p < 0.05) differentially expressed genes (DEGs), which were analysed into up-regulated and down-regulated genes. Further, DEGs were functionally characterized by the Gene Ontology annotations and the Kyoto Encyclopedia of Genes and Genomes pathways. Overall, some of the up-regulated genes in resistant genotypes were disease resistance proteins such as CC-NBS-LRR resistance protein, ankyrin repeat family protein, cytochrome P450, leucine-rich repeat family protein/protein kinase family, and MYB transcription factor. Sequence diversity analysis based on 38 peptide sequences representing 18 genes showed distinct variation and the presence of three motifs in 15 amino acid sequences. Selected genes were also validated by real-time quantitative polymerase chain reaction analysis. Interestingly, gene expression markers were developed for late blight resistant genotypes. Our study elucidates genes involved in imparting late blight resistance in potato, which will be beneficial for its management strategies in the future.

Development of near homozygous lines for diploid hybrid TPS breeding in potatoes.

Diploid inbred-based F1 hybrid True Potato Seed (DHTPS) breeding is a novel technique to transform potato breeding and cultivation across the globe. Significant efforts are being made to identify elite diploids, dihaploids and develop diploid inbred lines for heterosis exploitation in potatoes. Self-incompatibility is the first obstacle for developing inbred lines in diploid potatoes, which necessitates the introgression of a dominant S locus inhibitor gene (Sli) for switching self-incompatibility to self-compatibility. We evaluated a set of 357 diploid clones in different selfing generations for self-compatibility and degree of homozygosity using Kompetitive Allele Specific PCR (KASP) Single Nucleotide Polymorphism (SNP) markers. A subset of 10 KASP markers of the Sli candidate region on chromosome 12 showed an association with the phenotype for self-compatibility. The results revealed that the selected 10 KASP markers for the Sli gene genotype could be deployed for high throughput rapid screening of self-compatibility in diploid populations and to identify new sources of self-compatibility. The homozygosity assessed through 99 KASP markers distributed across all the chromosomes of the potato genome was 20-78 % in founder diploid clones, while different selfing generations, i.e., S0, S1, S2 and S3 observed 36.1-80.4, 56.9-82.8, 59.5-85.4 and 73.7-87.8 % average homozygosity, respectively. The diploid plants with ∼80 % homozygosity were also observed in the first selfing generation, which inferred that homozygosity assessment in the early generations itself could identify the best plants with high homozygosity to speed up the generation of diploid inbred lines.

Transcriptome profiling and characterization of genes associated with tuberization under high temperature in aeroponics in potato cv. Kufri Anand.

BACKGROUND: High temperature stress is an important abiotic factor, which affects tuberization and ultimately causes heavy yield reduction in potato. OBJECTIVES: Identification and characterization of genes associated with tuberization under high temperature stress is essential for future management through biotechnology. METHODOLOGY: Two contrasting potato varieties Kufri Anand (profuse tuber-bearing) versus Kufri Frysona (very less/scanty tuber-bearing, control) were cultivated in aeroponics under high temperature stress, and transcriptomes were analyzed. RESULTS: Potato cv. Kufri Anand was found superior over control (Kufri Frysona) for tuber yield and its component traits along with root morphology under aeroponics. Transcriptomes of tuber and leaf tissues were analyzed. Statistically significant (p < 0.05) differentially expressed genes (DEGs) were categorised into up-regulated (> 2 log2 fold change, FC) and down-regulated (< -2 log2 FC) genes. DEGs were annotated by gene ontology and KEGG pathways. A few selected up-regulated genes of both tissues were identified, and phylogeny tree and motif analysis were analysed based on 36 peptide sequences representing 15 selected DEGs in this study. Further, gene expression markers were developed and validated by real time qPCR analysis for the identification of high temperature tolerant genotypes. CONCLUSION: A few key genes associated in tuberization under high temperature conditions were heat shock proteins (e.g. 18.5 kDa class I heat shock protein), sugar metabolism (e.g. glucosyltransferase), transcription factor (e.g. WRKY), and phytohormones (e.g. auxin-induced beta-glucosidase). Our study provides an overview of key genes involved in tuberization under high temperature stress in potato cv. Kufri Anand under aeroponics.

Screening of wild species and transcriptome profiling to identify differentially regulated genes in response to late blight resistance in potato.

Late blight (Phytophthora infestans) is a serious disease of potatoes. The aim of this study was to screen wild potato species and identify differentially expressed genes (DEGs) associated with late blight resistance. Wild potato species such as PIN45 (Solanum pinnatisectum), CPH62 (Solanum cardiophyllum), JAM07 (Solanum jamesii), MCD24 (Solanum microdontum), PLD47 (Solanum polyadenium), and cv. Kufri Bahar (control) were tested by artificial inoculation of P. infestans under controlled conditions. Transcriptomes of the leaf tissues (96 h post-inoculation) were sequenced using the Illumina platform. Statistically significant (p < 0.05) DEGs were analyzed in wild species by comparison with the control, and upregulated (>2 log2 fold change, FC) and downregulated (<-2 log2 FC) genes were identified. DEGs were functionally characterized with Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Selected genes were validated by real-time PCR analysis to confirm RNA-seq results. We identified some upregulated genes associated with late blight resistance in wild species such as cytochrome P450, proline-rich protein, MYB transcription factor MYB139, ankyrin repeat-containing protein, and LRR receptor-like serine/threonine-protein kinase in PIN45; glucosyltransferase, fructose-bisphosphate aldolase, and phytophthora-inhibited protease 1 in CPH62; steroid binding protein and cysteine proteinase 3 in JAM07; glycine-rich cell wall structural protein 1 and RING finger protein in MCD24; and cysteine proteinase 3 and major latex protein in PLD47. On the other hand, downregulated genes in these species were snakin-2 and WRKY transcription factor 3 in PIN45; lichenase and phenylalanine ammonia-lyase 1 in CPH62; metallothionein and LRR receptor-like serine/threonine-protein kinase in JAM07; UDP-glucoronosyl/UDP-glucosyl transferase family protein and steroid binding protein in MCD24; and cytoplasmic small heat shock protein class I and phosphatase PLD47. Our study identified highly resistant wild potato species and underlying genes such as disease resistance, stress response, phytohormones, and transcription factors (e.g., MYB, WRKY, AP2/ERF, and AN1) associated with late blight resistance in wild potato species.

Root system architecture for abiotic stress tolerance in potato: Lessons from plants.

The root is an important plant organ, which uptakes nutrients and water from the soil, and provides anchorage for the plant. Abiotic stresses like heat, drought, nutrients, salinity, and cold are the major problems of potato cultivation. Substantial research advances have been achieved in cereals and model plants on root system architecture (RSA), and so root ideotype (e.g., maize) have been developed for efficient nutrient capture to enhance nutrient use efficiency along with genes regulating root architecture in plants. However, limited work is available on potatoes, with a few illustrations on root morphology in drought and nitrogen stress. The role of root architecture in potatoes has been investigated to some extent under heat, drought, and nitrogen stresses. Hence, this mini-review aims to update knowledge and prospects of strengthening RSA research by applying multi-disciplinary physiological, biochemical, and molecular approaches to abiotic stress tolerance to potatoes with lessons learned from model plants, cereals, and other plants.

Germplasm, Breeding, and Genomics in Potato Improvement of Biotic and Abiotic Stresses Tolerance.

Potato is one of the most important food crops in the world. Late blight, viruses, soil and tuber-borne diseases, insect-pests mainly aphids, whiteflies, and potato tuber moths are the major biotic stresses affecting potato production. Potato is an irrigated and highly fertilizer-responsive crop, and therefore, heat, drought, and nutrient stresses are the key abiotic stresses. The genus Solanum is a reservoir of genetic diversity, however, a little fraction of total diversity has been utilized in potato breeding. The conventional breeding has contributed significantly to the development of potato varieties. In recent years, a tremendous progress has been achieved in the sequencing technologies from short-reads to long-reads sequence data, genomes of Solanum species (i.e., pan-genomics), bioinformatics and multi-omics platforms such as genomics, transcriptomics, proteomics, metabolomics, ionomics, and phenomics. As such, genome editing has been extensively explored as a next-generation breeding tool. With the available high-throughput genotyping facilities and tetraploid allele calling softwares, genomic selection would be a reality in potato in the near future. This mini-review covers an update on germplasm, breeding, and genomics in potato improvement for biotic and abiotic stress tolerance.

Resistance Evaluation for Native Potato Accessions against Late Blight Disease and Potato Cyst Nematodes by Molecular Markers and Phenotypic Screening in India.

The potato originated in southern Peru and north-western Bolivia (South America). However, native accessions have also been cultivated in India for many years. Late blight, caused by the fungus Phytophthora infestans, is the most devastating potato disease, while potato cyst nematode (Globodera spp.) (PCN) is another economically significant quarantine-requiring pest in India. In this study, we have generated a new Indian native collection of 94 potato accessions collected from different parts India. These accessions were screened against late blight and potato cyst nematode resistance by using gene-based molecular markers and phenotypic screening methods. Marker assisted selection using R1 gene-specific marker CosA210 revealed a late blight resistance gene in 11 accessions. PCN resistance bands were found in 3 accessions with marker TG689141, 5 accessions with marker 57R452, and 1 accession having Gro1-4-1602 marker for G. rostochiensis (Ro1,4), while 64 accessions amplified marker HC276 indicating G. pallida (Pa2,3) resistance gene (GpaVvrn QTL). On the other hand, phenotypic screening against late blight resistance under natural epiphytic conditions (hot-spot) revealed three accessions with high resistance, while others were resistant (1 accession), moderately resistant (5 accessions), susceptible (29 accessions), and highly susceptible (56 accessions). For G. rostochiensis (golden cyst nematode) and G. pallida (white cyst nematode) resistance, accessions were grouped into highly resistant (3, 3), resistant (0, 2), moderately resistant (6, 29), susceptible (32, 30), and highly susceptible (53, 30), respectively, for the two PCN species. Collectively, we identified promising accessions with high resistance to late blight (JG-1, Kanpuria Safed, and Rangpuria), and also highly resistant to both Globodera species (Garlentic, Jeevan Jyoti, and JG-1). Our findings suggested that these accessions would be useful for late blight and PCN resistance breeding, as well as future molecular studies in potatoes.

A new record of Asia II 5 genetic group of Bemisia tabaci (Gennadius) in the major potato growing areas of India and its relationship with tomato leaf curl New Delhi virus infecting potato.

The whitefly, Bemisia tabaci (Gennadius), is responsible for significant yield losses in many crops, including potato, by sucking the phloem sap and transmitting a number of plant viruses. B. tabaci is a complex of cryptic species which is commonly designated as genetic groups. The B. tabaci genetic groups differ biologically with respect to host plant preference, insecticidal resistance, reproduction capacity, and ability to transmit begomoviruses. Therefore, understanding genetic variation among populations is important for establishing crop-specific distribution profile and management. We sequenced the mitochondrial cytochrome oxidase I (mtCOI) gene of B. tabaci collected from major potato growing areas of India. BLAST analysis of the 24 mtCOI sequences with reference Gene Bank sequences revealed four B. tabaci genetic groups prevailing in this region. mtCOI analysis exhibited the presence of Asia II 1, Asia II 5, Asia 1, and MEAM1 B. tabaci genetic groups. Our study highlighted that a new genetic group Asia II 5 has been detected in Indo-Gangetic Plains. Further virus-vector relationship study of ToLCNDV with Asia II 5 B. tabaci revealed that females are efficient vector of this virus as compared to males. This behavior of females might be due to their ability to acquire more virus titer than males. This study will help in better understanding of whitefly genetic group mediated virus diseases.

Physiological and genome-wide RNA-sequencing analyses identify candidate genes in a nitrogen-use efficient potato cv. Kufri Gaurav.

Nitrogen (N) is an important nutrient for plant growth. However, its excess application leads to environmental damage. Hence, improving nitrogen use efficiency (NUE) of plant is one of the plausible options to solve the problems. Aim of this study was to identify candidate genes involved in enhancing NUE in potato cv. Kufri Gaurav (N efficient). Plants were grown in aeroponic with two contrasting N regimes (low N: 0.75 mM, and high N: 7.5 mM). Higher NUE in Kufri Gaurav was observed in low N based on the parameters like NUE, NUpE (N uptake efficiency), NUtE (N utilization efficiency) and AgNUE (agronomic NUE). Further, global gene expression profiles in root, leaf and stolon tissues were analyzed by RNA-sequencing using Ion Proton™ System. Quality data (≥Q20) of 2.04-2.73 Gb per sample were mapped with the potato genome. Statistically significant (P ≤ 0.05) differentially expressed genes (DEGs) were identified such as 176 (up-regulated) and 30 (down-regulated) in leaves, 39 (up-regulated) and 105 (down-regulated) in roots, and 81 (up-regulated) and 694 (down-regulated) in stolons. The gene ontology (GO) terms like metabolic process, cellular process and catalytic activity were predominant. Our RT-qPCR analysis confirmed the gene expression profiles of RNA-seq. Overall, we identified candidate genes associated with improving NUE such as superoxide dismutase, GDSL esterase lipase, probable phosphatase 2C, high affinity nitrate transporters, sugar transporter, proline rich proteins, transcription factors (VQ motif, SPX domain, bHLH) etc. Our findings suggest that these candidate genes probably play crucial roles in enhancing NUE in potato.

Genome-wide identification and characterization of microRNAs by small RNA sequencing for low nitrogen stress in potato.

Nitrogen is an important nutrient for plant growth and tuber quality of potato. Since potato crop requires high dose of N, improving nitrogen use efficiency (NUE) of plant is an inevitable approach to minimize N fertilization. The aim of this study was to identify and characterize microRNAs (miRNAs) by small RNA sequencing in potato plants grown in aeroponic under two contrasting N (high and low) regimes. A total of 119 conserved miRNAs belonging to 41 miRNAs families, and 1002 putative novel miRNAs were identified. From total, 52 and 54 conserved miRNAs, and 404 and 628 putative novel miRNAs were differentially expressed in roots and shoots, respectively under low N stress. Of total 34,135 predicted targets, the gene ontology (GO) analysis indicated that maximum targets belong to biological process followed by molecular function and cellular component. Eexpression levels of the selected miRNAs and targets were validated by real time-quantitative polymerase chain reaction (RT-qPCR) analysis. Two predicted targets of potential miRNAs (miR397 and miR398) were validated by 5' RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). In general, predicted targets are associated with stress-related, kinase, transporters and transcription factors such as universal stress protein, heat shock protein, salt-tolerance protein, calmodulin binding protein, serine-threonine protein kinsae, Cdk10/11- cyclin dependent kinase, amino acid transporter, nitrate transporter, sugar transporter, transcription factor, F-box family protein, and zinc finger protein etc. Our study highlights that miR397 and miR398 play crucial role in potato during low N stress management. Moreover, study provides insights to modulate miRNAs and their predicted targets to develop N-use efficient potato using transgenic/genome-editing tools in future.

Transcriptome analysis of potato shoots, roots and stolons under nitrogen stress.

Potato crop requires high dose of nitrogen (N) to produce high tuber yield. Excessive application of N causes environmental pollution and increases cost of production. Hence, knowledge about genes and regulatory elements is essential to strengthen research on N metabolism in this crop. In this study, we analysed transcriptomes (RNA-seq) in potato tissues (shoot, root and stolon) collected from plants grown in aeroponic culture under controlled conditions with varied N supplies i.e. low N (0.2 milli molar N) and high N (4 milli molar N). High quality data ranging between 3.25 to 4.93 Gb per sample were generated using Illumina NextSeq500 that resulted in 83.60-86.50% mapping of the reads to the reference potato genome. Differentially expressed genes (DEGs) were observed in the tissues based on statistically significance (p ≤ 0.05) and up-regulation with ≥ 2 log2 fold change (FC) and down-regulation with ≤ -2 log2 FC values. In shoots, of total 19730 DEGs, 761 up-regulated and 280 down-regulated significant DEGs were identified. Of total 20736 DEGs in roots, 572 (up-regulated) and 292 (down-regulated) were significant DEGs. In stolons, of total 21494 DEG, 688 and 230 DEGs were significantly up-regulated and down-regulated, respectively. Venn diagram analysis showed tissue specific and common genes. The DEGs were functionally assigned with the GO terms, in which molecular function domain was predominant in all the tissues. Further, DEGs were classified into 24 KEGG pathways, in which 5385, 5572 and 5594 DEGs were annotated in shoots, roots and stolons, respectively. The RT-qPCR analysis validated gene expression of RNA-seq data for selected genes. We identified a few potential DEGs responsive to N deficiency in potato such as glutaredoxin, Myb-like DNA-binding protein, WRKY transcription factor 16 and FLOWERING LOCUS T in shoots; high-affinity nitrate transporter, protein phosphatase-2c, glutaredoxin family protein, malate synthase, CLE7, 2-oxoglutarate-dependent dioxygenase and transcription factor in roots; and glucose-6-phosphate/phosphate translocator 2, BTB/POZ domain-containing protein, F-box family protein and aquaporin TIP1;3 in stolons, and many genes of unknown function. Our study highlights that these potential genes play very crucial roles in N stress tolerance, which could be useful in augmenting research on N metabolism in potato.